Abstract Antimicrobial resistance has become one of the greatest threats to public health, with rising resistance to carbapenems being a particular concern. Cefiderocol is a novel catechol-substituted siderophore cephalosporin which is currently undergoing late-stage clinical development. It is actively transported across the Gram-negative outer membrane and is stable against all classes of beta-lactamases, resulting in potent activity across Gram-negative bacterial species. While rare cefiderocol-resistant strains have been reported in surveillance studies, the mechanisms underlying cefiderocol resistance are largely unknown. When we tested susceptibility of our collection of carbapenem-resistant Enterobacteriaceae strains to cefiderocol, we encountered strains with MICs of 1 mg/L or higher, and sometimes greater than 4 mg/L, which is the susceptibility breakpoint for this agent. Among KPC-producing, carbapenem-resistant K. pneumoniae strains, those that produced KPC with certain amino acid substitutions associated with resistance to ceftazidime- avibactam, such as D179Y, V240G and D179Y/T243M, showed 2 to 8-fold higher cefiderocol MICs compared with strains producing wild-type KPC-3. These differences were reproduced in isogenic E. coli laboratory strains. In addition, several non-KPC-producing, carbapenem-resistant Enterobacter spp. clinical strains also showed resistance to cefiderocol. In one of these strains that showed cefiderocol MIC of >16 mg/L, we identified chromosomal AmpC beta-lactamase that contained a two amino acid deletion in the R2 loop structure. This variant AmpC conferred reduced cefiderocol susceptibility when expressed in E. coli, and was confirmed to hydrolyze cefiderocol. The structures of apo-enzyme and the enzyme?drug complex revealed the role of the deletion in extending the loop in the alpha-helix structure allowing for the accommodation of the bulky R2 side chain of cefiderocol. These preliminary findings have led us to hypothesize that specific structural changes in broad-spectrum beta-lactamases allow them to accommodate and hydrolyze cefiderocol thereby conferring reduced susceptibility or frank resistance to cefiderocol, and that some of these changes also impact hydrolysis of ceftazidime and/or binding of avibactam. To address these hypotheses, we propose the following Specific Aims: (i) To elucidate the kinetics and structure of cefiderocol-hydrolyzing beta- lactamases, and (ii) To characterize de novo variant beta-lactamases and non-enzymatic resistance mechanisms that emerge upon exposure to cefiderocol. With its unparalleled spectrum of activity across Gram- negative species, cefiderocol is likely to become a crucially important agent in the treatment of carbapenem- resistant Gram-negative infections, but information regarding the mechanisms of resistance and the risk of their emergence is non-existent. Our proposal will address these key questions to optimize use of cefiderocol in the clinic. Furthermore, insights into the structure-activity relationship of substrate-binding sites of cefiderocol-hydrolyzing beta-lactamases will inform designing of the next-generation cephalosporins.